Ulrich G. Mueller

AFLP genotyping and fingerprinting

Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP methods rapidly generate hundreds of highly replicable markers from DNA of any organism; thus, they allow high-resolution genotyping of fingerprinting quality. The time and cost efficiency, replicability and resolution of AFLPs are superior or equal to those of other markers [allozymes, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), microsatellites], except that AFLP methods primarily generate dominant rather than co-dominant markers. Because of their high replicability and ease of use, AFLP markers have emerged as a major new type of genetic marker with broad application in systematics, pathotyping, population genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.

The Evolution of Cooperation

Darwin recognized that natural selection could not favor a trait in one species solely for the benefit of another species. The modern, selfish‐gene view of the world suggests that cooperation between individuals, whether of the same species or different species, should be especially vulnerable to the evolution of noncooperators. Yet, cooperation is prevalent in nature both within and between species. What special circumstances or mechanisms thus favor cooperation? Currently, evolutionary biology offers a set of disparate explanations, and a general framework for this breadth of models has not emerged. Here, we offer a tripartite structure that links previously disconnected views of cooperation. We distinguish three general models by which cooperation can evolve and be maintained: (i) directed reciprocation—cooperation with individuals who give in return; (ii) shared genes—cooperation with relatives (e.g., kin selection); and (iii) byproduct benefits—cooperation as an incidental consequence of selfish action. Each general model is further subdivided. Several renowned examples of cooperation that have lacked explanation until recently—plant‐rhizobium symbioses and bacteria‐squid light organs—fit squarely within this framework. Natural systems of cooperation often involve more than one model, and a fruitful direction for future research is to understand how these models interact to maintain cooperation in the long term.

The evolution of mutualisms: exploring the paths between conflict and cooperation

Mutualisms are of fundamental importance in all ecosystems but their very existence poses a series of challenging evolutionary questions. Recently, the application of molecular analyses combined with theoretical advances have transformed our understanding of many specific systems, thereby contributing to the possibility of a more general understanding of the factors that influence mutualisms.

Evolutionary history of the symbiosis between fungus-growing ants and their fungi

The evolutionary history of the symbiosis between fungus-growing ants (Attini) and their fungi was elucidated by comparing phylogenies of both symbionts. The fungal phylogeny based on cladistic analyses of nuclear 28S ribosomal DNA indicates that, in contrast with the monophyly of the ants, the attine fungi are polyphyletic. Most cultivated fungi belong to the basidiomycete family Lepiotaceae; however, one ant genus, Apterostigma, has acquired a distantly related basidiomycete lineage. Phylogenetic patterns suggest that some primitive attines may have repeatedly acquired lepiotaceous symbionts. In contrast, the most derived attines have clonally propagated the same fungal lineage for at least 23 million years.

THE ORIGIN OF THE ATTINE ANT-FUNGUS MUTUALISM

Cultivation of fungus for food originated about 45-65 million years ago in the ancestor of fungus-growing ants (Formicidae, tribe Attini), representing an evolutionary transition from the life of a hunter-gatherer of arthropod prey, nectar, and other plant juices, to the life of a farmer subsisting on cultivated fungi. Seven hypotheses have been suggested for the origin of attine fungiculture, each differing with respect to the substrate used by the ancestral attine ants for fungal cultivation. Phylogenetic information on the cultivated fungi, in conjunction with information on the nesting biology of extant attine ants and their presumed closest relatives, reveal that the attine ancestors probably did not encounter their cultivars-to-be in seed stores (von Ihering 1894), in rotting wood (Forel 1902), as mycorrhizae (Garling 1979), on arthropod corpses (von Ihering 1894) or ant faeces in nest middens (Wheeler 1907). Rather, the attine ant-fungus mutualism probably arose from adventitious interactions with fungi that grew on walls of nests built in leaf litter (Emery 1899), or from a system of fungal myrmecochory in which specialized fungi relied on ants for dispersal (Bailey 1920) and in which the ants fortuitously vectored these fungi from parent to offspring nests prior to a true fungicultural stage.

Bacterial Diversity in Solenopsis invicta and Solenopsis geminata Ant Colonies Characterized by 16S amplicon 454 Pyrosequencing

Social insects harbor diverse assemblages of bacterial microbes, which may play a crucial role in the success or failure of biological invasions. The invasive fire ant Solenopsis invicta (Formicidae, Hymenoptera) is a model system for understanding the dynamics of invasive social insects and their biological control. However, little is known about microbes as biotic factors influencing the success or failure of ant invasions. This pilot study is the first attempt to characterize and compare microbial communities associated with the introduced S. invicta and the native Solenopsis geminata in the USA. Using 16S amplicon 454 pyrosequencing, bacterial communities of workers, brood, and soil from nest walls were compared between neighboring S. invicta and S. geminata colonies at Brackenridge Field Laboratory, Austin, Texas, with the aim of identifying potential pathogenic, commensal, or mutualistic microbial associates. Two samples of S. geminata workers showed high counts of Spiroplasma bacteria, a known pathogen or mutualist of other insects. A subsequent analysis using PCR and sequencing confirmed the presence of Spiroplasma in additional colonies of both Solenopsis species. Wolbachia was found in one alate sample of S. geminata, while one brood sample of S. invicta had a high count of Lactococcus. As expected, ant samples from both species showed much lower microbial diversity than the surrounding soil. Both ant species had similar overall bacterial diversities, although little overlap in specific microbes. To properly characterize a single bacterial community associated with a Solenopsis ant sample, rarefaction analyses indicate that it is necessary to obtain 5,000–10,000 sequences. Overall, 16S amplicon 454 pyrosequencing appears to be a cost-effective approach to screen whole microbial diversity associated with invasive ant species.

Ant versus Fungus versus Mutualism: Ant‐Cultivar Conflict and the Deconstruction of the Attine Ant‐Fungus Symbiosis

A century of research on fungus‐growing ants (Attini, Formicidae) has ignored the cultivated fungi as passive domesticates and viewed the attine fungicultural symbiosis as an integrated unit dominated by the evolutionary interests of the ant farmers. This article takes a different perspective and explores first the evolutionary interests and leverages of the fungal cultivars, then dissects eight potential evolutionary conflicts between ants and cultivars. Three types of ant‐cultivar conflict are examined in depth. First, ant‐cultivar conflict over the ant sex ratio is predicted because the cultivars are dispersed by female foundresses but not by males; cultivars thus may be selected to bias the ant sex ratio toward females. Second, ant‐cultivar conflict over fungal sexual reproduction exists if the fungi are able to escape from the symbiosis and live independently, as is implied by phylogenetic analyses of the fungi; this conflict is exacerbated in colonies that experience queen death or senescence. A literature review reveals that sexual fruiting of attine cultivars is more common than has been traditionally realized and often occurs in moribund colonies. Third, the routine transplanting of fungal mycelium by ants could generate, through sensory‐biased symbiont choice, selection favoring fungal features that increase the likelihood of transplantation within nests (symbiont drive) but that are detrimental to the survival of the whole colony. A balanced perspective incorporating both ant and fungal interests emerges as a more appropriate framework than the traditional myrmicocentric perspective. Indeed, the attine symbiosis offers unique experimental opportunities (cultivar switch experiments) to unravel the evolutionary dynamics of conflict and cooperation between ant and fungal partners.

Fine-scale genetic pattern and evidence for sex-biased dispersal in the túngara frog, Physalaemus pustulosus

Túngara frogs (Physalaemus pustulosus) are a model system for sexual selection and communication. Population dynamics and gene flow are of major interest in this species because they influence speciation processes and microevolution, and could consequently provide a deeper understanding of the evolutionary processes involved in mate recognition. Although earlier studies have documented genetic variation across the species’ range, attempts to investigate dispersal on a local level have been limited to mark–recapture studies. These behavioural studies indicated high mobility at a scale of several hundred metres. In this study we used seven highly polymorphic microsatellite loci to investigate fine‐scaled genetic variation in the túngara frog. We analysed the influence of geographical distance on observed genetic patterns, examined the influence of a river on gene flow, and tested for sex‐biased dispersal. Data for 668 individuals from 17 populations ranging in distance from 0.26 to 11.8 km revealed significant levels of genetic differentiation among populations. Genetic differentiation was significantly correlated with geographic distance. A river acted as an efficient barrier to gene flow. Several tests of sex‐biased dispersal were conducted. Most of them showed no difference between the sexes, but variance of Assignment Indices exhibited a statistically significant male bias in dispersal.

COEVOLUTION BETWEEN ATTINE ANTS AND ACTINOMYCETE BACTERIA : A REEVALUATION

Abstract We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution—reciprocal modification—therefore remains to be established for the attine ant-actinomycete mutualism.

Microfungal 'Weeds' in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.